Opioid peptides are suggested to serve as regulatory signals between the neuroendocrine and immune systems as well as among immune system cells. Opioid peptides (i.e. enkephalins and endorphins) affect a number of immune system functions in vitro (e.g. antibody production, cell proliferation, cytotoxicity, lymphokine production, and chemotaxis) and some immunocompetent cell types synthesize and release enkephalins and beta-endorphin in response to inflammatory stimuli. Opioid modulation of severely compromised immune systems (e.g. acquired immunodeficiency syndrome and cancer) has been reported. Extracellularly oriented cell surface peptidases (ectopeptidases) play a key role in metabolism and inactivation of opioid peptides in several tissues. We postulate that ectopeptidases are also important in the immune system in modulating local opioid concentrations and the spectrum of inactive and active opioid peptides produced. Some cell surface antigens used to characterize hemopoietic cells have recently been identified as ectopeptidases (e.g. CALLA/CD10 as neutral endopeptidase and CD13 as aminopeptidase N). Our broad goal is to better understand the regulatory role of ectopeptidases in the physiological response of immunocompetent cells to opioid peptides. Cell amalgams are not suitable experimental models to identify specific mechanisms involved in modulating opioid effects on immunocompetent cells. Our approach has been to use highly purified, well-characterized subsets of immune cells (CD4+ and CD8+ T cells and macrophages) as well as immortalized cell lines to identify the peptidases which metabolize opioid peptides in immunocompetent cells and the role of these peptidases in regulating specific opioid modulated cell functions. Our first specific aim is to investigate the role of aminopeptidase N in regulating enkephalin modulated T cell and macrophage functions by using anti-sense techniques to specifically suppress its expression. Our current work has identified aminopeptidase N as the predominant enkephalin metabolizing ectopeptidase in both macrophages and T cells. The second specific aim is to isolate and characterize a second enkephalin metabolizing aminopeptidase which we have identified in macrophages and which is distinct from other known aminopeptidases. The third aim is to isolate and characterize a unique T cell ectopeptidase which we have found which metabolizes beta-endorphin to smaller peptides with immunomodulating activities distinct from the parent peptide. A number of strategies will be used to investigate the physiologic function of these two new peptidases, including examination of metabolites for immunomodulatory activity, effects of specific inhibitors of the peptidases, and specific suppression of the peptidase activities with anti-sense techniques upon obtaining sequence information. Successful completion of these studies will enhance our knowledge of opioid peptide regulation of the immune system and provide a solid biochemical foundation for future therapeutic approaches to many immune system dysfunctions.